Development of Mycobacterium tuberculosis DNA gyrase as a target for antibacterial chemotherapy

291 Pages·2010·17.49 MB·English

Development of Mycobacterium
tuberculosis DNA gyrase
as a target for antibacterial
chemotherapy

Shantanu Karkare

John Innes Centre

September 2010
This thesis is submitted in partial fulfilment of the requirements of the
degree of Doctor of Philosophy at the University of East Anglia.
© This copy of the thesis has been supplied on condition that anyone who
consults it is understood to recognise that its copyright rests with the author and
that no quotation from the thesis, nor any information derived therefrom, may
be published without the authors prior, written consent.
1
A
BSTRACT

Bacterial DNA gyrase is one of the proven targets for antibacterial
chemotherapy. It is a type II DNA topoisomerase found in all bacteria. Most of
our current information is related to the enzyme from Escherichia coli (E. coli),
a Gram-negative bacterium, with limited information about the gyrase from
Mycobacterium tuberculosis (M. tuberculosis), a Gram-positive bacterium. The
emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-
resistant tuberculosis (XDR-TB) with no new classes of drugs has posed a great
challenge for the effective and short-term treatment of tuberculosis. Thus, it has
become important to understand and investigate established drug targets, such
as DNA gyrase, in M. tuberculosis. Although there are similarities between the
gyrases from these two bacteria, there are key differences, which can be
potentially exploited for identifying new drugs for tuberculosis. This work
describes the identification of a putative Ca2+-binding site in M. tuberculosis
GyrA and discovery of naphthoquinones as novel inhibitors of DNA gyrase.
In the absence of a complete crystal structure for M. tuberculosis GyrA, a
homology model was constructed for biochemical and site-directed mutagenesis
experiments involving the putative Ca2+-binding site. These experiments
indicate that Ca2+ has a potential regulatory role in M. tuberculosis. Virtual
screening was performed to identify novel inhibitors targeting the Ca2+-binding
site.
Diospyrin, a naphthoquinone was identified as a potent inhibitor of DNA
gyrase. This work has demonstrated that diospyrin inhibits DNA gyrase with a
novel mechanism and that the GyrB N-terminal domain is the potential target
site. Docking studies were performed to predict the diospyrin-binding site in
GyrB.
2
T C
ABLE OF ONTENTS
ABSTRACT ................................................................................................................ 2
LIST OF FIGURES ...................................................................................................... 7
LIST OF TABLES ..................................................................................................... 14
ACKNOWLEDGEMENTS .......................................................................................... 17
Chapter 1: General Introduction................................................................................. 18
1.1 Tuberculosis ..................................................................................................... 18
1.2 DNA topology .................................................................................................. 22
1.3 Biological significance of DNA topology........................................................ 24
1.4 DNA topoisomerases ....................................................................................... 25
1.4.1 Introduction ............................................................................................... 25
1.4.2 Type IA topoisomerases ............................................................................ 28
1.4.3 Structure of type IA topoisomerase........................................................... 30
1.4.4 Mechanism of action of type IA topoisomerase ....................................... 32
1.4.5 Type IB topoisomerases ............................................................................ 33
1.4.6 Mechanism of action of type IB topoisomerases ...................................... 35
1.4.7 Type II DNA topoisomerases.................................................................... 37
1.4.8 Structure of Type IIA topoisomerases ...................................................... 38
1.4.9 ATP hydrolysis by type II topoisomerases ............................................... 41
1.4.10 Mechanism of type IIA topoisomerases .................................................. 41
1.5 DNA gyrase ...................................................................................................... 44
1.5.1 Introduction ............................................................................................... 44
1.5.2 DNA gyrase strand passage mechanism ................................................... 49
1.5.3 DNA wrapping by DNA gyrase ................................................................ 49
1.5.4 DNA cleavage-religation reaction by DNA gyrase .................................. 50
1.5.5 ATP hydrolysis by DNA gyrase ............................................................... 52
1.5.6 DNA gyrase in eukaryotes ........................................................................ 54
1.6 Type IIB topoisomerases ................................................................................. 54
1.7 DNA gyrase inhibitors ..................................................................................... 56
1.7.1 Fluoroquinolones ...................................................................................... 57
1.7.2 Aminocoumarins ....................................................................................... 66
1.7.3 Simocyclinones ......................................................................................... 69
1.7.4 Cyclothialidine .......................................................................................... 71
1.7.5 GSK299423 ............................................................................................... 73
1.7.6 CcdB .......................................................................................................... 75
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1.7.7 Microcin B17 ............................................................................................ 76
1.8 Other proteinaceous inhibitors ......................................................................... 78
1.9 Aim of the project ............................................................................................ 80
Chapter 2: Materials & Methods ................................................................................ 81
2.1 Bacteriology ..................................................................................................... 81
2.1.1 E. coli and M. smegmatis strains ............................................................... 81
2.1.2 Media and antibiotics ................................................................................ 82
2.1.3 Preparation of electrocompetent M. smegmatis cells ................................ 83
2.1.4 Preparation of chemically competent E. coli cells .................................... 84
2.1.5 Transformation .......................................................................................... 84
2.2 Molecular biology methods.............................................................................. 85
2.2.1 Oligonucleotides ....................................................................................... 85
2.2.2 Preparation of supercoiled and relaxed pBR322* ..................................... 87
2.2.3 Plasmid DNA purification......................................................................... 88
2.2.4 DNA concentration determination ............................................................ 88
2.2.5 PCR and cloning ....................................................................................... 89
2.2.6 Site-directed mutagenesis.......................................................................... 90
2.2.7 RT-PCR (reverse-transcriptase) ................................................................ 92
2.2.8 Agarose gel electrophoresis ...................................................................... 92
2.2.9 DNA sequencing ....................................................................................... 93
2.3 Protein Methods ............................................................................................... 94
2.3.1 Purification of M. tuberculosis DNA gyrase and mutants ..................... 94
2.3.2 SDS-PAGE ................................................................................................ 95
2.3.3 Dialysis ...................................................................................................... 96
2.3.4 Protein concentration ................................................................................ 97
2.3.5 Protein concentration determination ......................................................... 97
2.3.6 Limited Proteolysis ................................................................................... 98
2.3.7 Electroblotting & Edman sequencing ....................................................... 98
2.3.8 Peptide mass fingerprinting by Orbitrap mass spectrometer .................... 99
2.3.9 Circular dichroism ................................................................................... 100
2.4 Enzyme assays ............................................................................................... 102
2.4.l DNA supercoiling, relaxation, and decatenation assays ......................... 102
2.4.2 DNA wrapping assay .............................................................................. 102
2.4.3 Promoter activity assay (β-galactosidase activity assay) ........................ 103
2.5 Biophysical techniques................................................................................... 104
2.5.1 ICP-AES .................................................................................................. 104
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2.5.2 Nano-ESI MS .......................................................................................... 106
2.5.3 BIAcore ................................................................................................... 107
2.5.4 Protein crystallography ........................................................................... 108
2.6 Bioinformatics software ................................................................................. 109
2.6.1 Clustal W and MUSCLE ......................................................................... 109
2.6.2 PSIPRED ................................................................................................. 110
2.6.3 PROSITE ................................................................................................ 110
2.6.4 Phyre and BioInfoBank MetaServer ....................................................... 110
2.6.5 Insight II .................................................................................................. 111
2.6.6 AutoDock Vina 1.0.2 .............................................................................. 111
2.6.7 Discovery Studio 2.5.5 ............................................................................ 112
Chapter 3: Unique features of M. tuberculosis DNA gyrase ................................ 113
3.1 Purification of M. tuberculosis DNA gyrase ................................................. 114
3.2 Bioinformatics analysis of M. tuberculosis DNA gyrase............................... 115
3.3 M. tuberculosis GyrB promoter analysis ....................................................... 116
3.3.1 RT-PCR for M. tuberculosis GyrB transcript ......................................... 117
3.3.2 Mass spectrometric analysis .................................................................... 120
3.3.3 GyrB N40∆ deletion mutant ................................................................... 121
3.3.4 Promoter analysis .................................................................................... 125
3.4 A putative Ca2+ -binding site in DNA gyrase ................................................. 130
3.4.1 EF-hand Ca2+ - binding domain .............................................................. 133
3.4.2 Model for the Ca2+- binding site ............................................................. 138
3.5 Conclusion ..................................................................................................... 144
Chapter4 : Investigation of a putative Ca2+-binding site in M. tuberculosis GyrA 148
4.1 Bacterial DNA gyrase Ca2+ selectivity ........................................................... 149
4.2 Ca2+-assisted Mg2+- dependent enzyme ......................................................... 153
4.3 SDM of the Ca2+-binding site ........................................................................ 163
4.4 Effect of EGTA on the activity of mutants .................................................... 174
4.5 Limited Proteolysis of GyrA .......................................................................... 177
4.6 Virtual screening at the Ca2+-binding site ...................................................... 183
4.6.1 Introduction ............................................................................................. 183
4.6.2 Docking-based virtual screening ............................................................. 184
4.7 Conclusion ..................................................................................................... 192
Chapter 5 : Identification and characterization of novel gyrase inhibitors ............. 196
5.1 Introduction .................................................................................................... 197
5.2 Quinolines ...................................................................................................... 198
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5.3 Aaptamines and marine extracts .................................................................... 201
5.4 Naphthoquinones ........................................................................................... 205
5.5 Targets for naphthoquinones .......................................................................... 208
5.6 Naphthoquinones inhibits DNA gyrase ......................................................... 211
5.7 E. coli Inhibitory activity ............................................................................... 219
5.8 Diospyrin binding site ................................................................................... 220
5.8.1 M. tuberculosis GyrB-diospyrin complex ............................................... 221
5.8.2 E. coli GyrB43-diospyrin complex ......................................................... 225
5.9 Limited Proteolysis ........................................................................................ 229
5.10 Surface plasmon resonance .......................................................................... 233
5.11 ATP competition assay ................................................................................ 239
5.12 M. tuberculosis GyrB43- diospyrin complex ............................................... 241
5.13 Discussion .................................................................................................... 247
Chapter 6: General Discussion ................................................................................. 252
6.1 Introduction .................................................................................................... 252
6.2 M. tuberculosis GyrB promoter analysis ....................................................... 253
6.3 Putative Ca2+-binding site .............................................................................. 254
6.4 NovelDNA gyrase inhibitors ......................................................................... 261

APPENDIX ................................................................................................................. 265
Crystallisation trials ............................................................................................. 266
ABBREVIATIONS....................................................................................................... 269
REFERENCES ............................................................................................................ 270

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IST OF IGURES
Figure 1.1: M. tuberculosis stained with Zein-Neelsen stain ..................................... 18
Figure 1.2: A global map showing the estimated TB incidence rates by country in
2007. ........................................................................................................................... 20
Figure 1.3 : Mechanism of action of different TB drugs. .......................................... 21
Figure 1.4: Significance of DNA topoisomerase.. .................................................... 26
Figure 1.5: Classification of Topoisomerase based on structure and mechanism. .... 27
Figure 1.6: Domain alignment of type IA topoisomerase showing the structural
diversity. ..................................................................................................................... 30
Figure 1.7: Structure of E. coli topo III and Archaeoglobus fulgidus reverse gyrase.
.................................................................................................................................... 31
Figure 1.8: General mechanism of action of type I topoisomerase............................ 33
Figure 1.9: Detailed mechanism of human topoisomerase I.. ................................... 35
Figure 1.10: Structure of human topoI. ..................................................................... 36
Figure 1.11: Reactions catalysed by typeII topoisomerases ..................................... 37
Figure 1.12: Domain organisation of type IIA topoisomerases ................................. 39
Figure 1.13: Conserved functional domains in type II topoisomerases. .................... 40
Figure 1.14: Two-gate mechanism for type II A topoisomerase ............................... 42
Figure 1.15: Crystal structures of different domains of E. coli DNA gyrase............. 43
Figure 1.16: Electrostatic map of the GyrB' dimer and DNA duplex ........................ 47
Figure 1.17: Crystal structures of different domains of M. tuberculosis DNA gyrase
.................................................................................................................................... 48
Figure 1.18: Crystal structures of GyrA C-terminal domain. .................................... 50
Figure 1.19: The two-metal ion mechanism for cleavage-religation reaction ........... 51
Figure 1.20: Interaction between GyrB24 and ATP showing hydrogen-bonds with
different residues. ....................................................................................................... 52
Figure 1.21: Crystal structure of apo topo VI ............................................................ 55
Figure 1.22 : The DNA gyrase reaction and sites of action of gyrase inhibitors. ...... 56
Figure 1.23: Structures of fluoroquinolones. ............................................................. 58
Figure 1.24: Crystal structure of the topo IV-DNA-moxifloxacin complex. ............. 59
Figure 1.25: Quinolone-topo IV cleavage complex. .................................................. 60
Figure 1.26: S. aureus DNA gyrase in complex with ciprofloxacin and DNA ......... 60
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Figure 1.27: MfpA, Qnr homologue found in M. tuberculosis. ................................. 65
Figure 1.28: The three main naturally-ocurring aminocoumarins: novobiocin,
clorobiocin, and coumermycin A . ............................................................................ 67
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Figure 1.29: Overlap of the novobiocin (green) and ADPNP (red) binding site at E.
coli GyrB N-terminus (GyrB24). ............................................................................... 68
Figure 1.30: Simocyclinone D8 binds to GyrA59. .................................................... 70
Figure 1.31: Cyclothialidine inhibits the ATPase activity of the GyrB subunit.. ...... 72
Figure 1.32: GSK299423 a novel DNA gyrase inhibitor. .......................................... 74
Figure 1.33: CcdB with multiple interacting partners: CcdA and DNA gyrase. ....... 76
Figure 1.34: MccB17 chemical structure and gene cluster. ....................................... 77
Figure 2.1: Flowchart for the protein refolding experiment. .................................... 97
Figure 2.2: CD spectra of poly-L-lysine at pH 11.1 and placental collagen ............ 101
Figure 2.3: Illustration of ICP-AES for the detection of metals or cations.............. 105
Figure 2.4: Electrospray ionisation process. ............................................................ 107
Figure 3.1: SDS-PAGE (12%) of DNA gyrase subunits purified by affinity
chromatography........................................................................................................ 114
Figure 3.2: Enzyme activity of purified M. tuberculosis DNA gyrase. ................... 115
Figure 3.3: Multiple sequence alignment of bacterial GyrB using ClustalW 1.83 .. 116
Figure 3.4: RT- PCR for M. tuberculosis GyrB transcripts ..................................... 119
Figure 3.5: Orbitrap analysis of M. tuberculosis GyrB ............................................ 121
Figure 3.6: Different fractions of GyrB N40∆ deletion mutant purified by HisTrap
.................................................................................................................................. 122
Figure 3.7: Effect of GyrB N40∆ on the decatenation activity of M. tuberculosis
DNA gyrase. ............................................................................................................. 123
Figure 3.8: Effect of GyrB N40∆ on the supercoiling and relaxation activity of M.
tuberculosis DNA gyrase ......................................................................................... 124
Figure 3.9: Promoters of M. tuberculosis DNA gyrase. .......................................... 125
Figure 3.10: Screen capture of Artemis analysis of gyr promoter elements ............ 126
Figure 3.11: Colonies of M. smegmatis mc2-155 obtained by transformation of the
cells with pSM-128, pSM-128-Met and pSM-128-Val plasmids ............................ 128
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Figure 3.12: β-galactosidase activity recorded from the crude extract of M.
smegmatis mc2-155 cells transformed with pSM-128, pSM-128-Val and pSM-128-
Met respectively. ...................................................................................................... 129
Figure 3.13: DNA gyrase is the only type II topoisomerase in M. tuberculosis ...... 131
Figure 3.14: Multiple sequence alignment of bacterial GyrA using ClustalW 1.83 131
Figure 3.15: Multiples sequence alignment for GyrA from B. melitensis, M.
tuberculosis and C. diphtheriae. .............................................................................. 132
Figure 3.16: The two major groups of EF-hands ..................................................... 134
Figure 3.17: The non-cannonical EF-hand Ca2+-binding sites ................................. 136
Figure 3.18: Illustration of differences between canonical and pseudo-EF hand .... 137
Figure 3.19: Sequence alignment for M. tuberculosis GyrA. .................................. 139
Figure 3.20: Homology model of M. tuberculosis GyrA Ca2+-binding site (MtGyrA-
1H71) based on the psychrophilic metalloprotease from Pseudomonas sp. (PDB
code: 1H71). ............................................................................................................. 141
Figure 3.21: Homology model of M. tuberculosis GyrA Ca2+-binding site (MtGyrA-
1BQB) based on Staphylococcus aureus metalloproteinase (PDB code: 1BQB).... 142
Figure 3.22: Difference between the 3-D environments of the GyrA Ca2+ -binding
site and the Staphylococcus aureus metalloproteinase (PDB id: 1BQB) template. . 144
Figure 3.23: The predicted Ca2+ -binding site in between the N- and C-terminal
domains of M. tuberculosis DNA gyrase. ................................................................ 147
Figure 4.1: E. coli and M. tuberculosis DNA gyrase supercoiling assay in the
presence of 6 mM Mg2+ and Ca2+. ............................................................................ 149
Figure 4.2: Supercoiling assay of M. tuberculosis and E. coli hybrid enzyme in the
presence of 6 mM Mg2+ and 6 mM Ca2+. ................................................................. 150
Figure 4.3: Effect of EGTA dialysis on the supercoiling and decatenation activity of
M. tuberculosis DNA gyrase. ................................................................................... 154
Figure 4.4: DNA relaxation assay in the presence and absence of 1 mM Ca2+ ....... 155
Figure 4.5: Time-course experiment to demonstrate the difference in the relaxation
activity of GyrA dialysed in the absence and presence of EGTA............................ 156
Figure 4.6: Experimental plan for protein refolding experiment. ............................ 157
Figure 4.7: Effect of protein refolding in the presence and absence of EGTA on the
supercoiling and relaxation activity of DNA gyrase.. .............................................. 162
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Figure 4.8: Effect of protein refolding in the presence and absence of EGTA on the
decatenation activity of DNA gyrase. ...................................................................... 163
Figure 4.9: Double mutation in the Ca2+ - binding site. ........................................... 164
Figure 4.10: Quadruple mutation in the Ca2+ - binding site. ................................... 164
Figure 4.11: Acidic amino-acids in MtGyrA-1H71 and MtGyrA-1BQB model that
were mutated to alanine in side-directed mutagenesis experiments. ....................... 165
Figure 4.12: Effect of double (GyrA E508A, D509A) and quadruple mutations. .. 167
Figure 4.13: Effect of quadruple mutation (GyrA D504A, E508A, D509A, E514A)
on the supercoiling activity under different assay conditions. ................................. 168
Figure 4.14: No significant effect of double (GyrA E508A, D509A) and quadruple
mutations (GyrA D504A, E508A, D509A, E514A) on the relaxation activity ....... 169
Figure 4.15: Decatenation activity of the wild-type and quadruple mutant. ............ 170
Figure 4.16: Far-UV (180-260 nm) CD spectra of wild-type GyrA and GyrA D504A,
E508A, D509A, E514A ........................................................................................... 171
Figure 4.17: The effect of quadruple mutation (D504A, E508A, D509A, and E514A)
on DNA wrapping around GyrA. ............................................................................. 173
Figure 4.18: Effect of EGTA on the supercoiling and relaxation activity of the
mutants. .................................................................................................................... 175
Figure 4.19: Far-UV (180-260 nm) CD spectra of wild-type GyrA (dialysed in 1
mM EGTA) and GyrA D504A, E508A, D509A, E514A (dialysed in 1 mM EGTA)
.................................................................................................................................. 176
Figure 4.20: Tryptic digest of the M. tuberculosis GyrA in the absence of Ca2+ and
in the presence of Ca2+ ............................................................................................ 178
Figure 4.21: Tryptic digest of the M. tuberculosis GyrB in the absence of Ca2+ and
in the presence of Ca2+. ............................................................................................ 179
Figure 4.22: Trypsin digestion profile of M. tuberculosis GyrA in the presence and
absence of Ca2+ at 30 min time ................................................................................. 180
Figure 4.23: Molecular weight estimation of the bands. .......................................... 181
Figure 4.24: The sequence coverage of band 2&8 (~47-kDa).. ............................... 182
Figure 4.25: Virtual screening of NCI diversity set II. ............................................ 185
Figure 4.26: List of 51 hit compounds from the virtual screening of NCI diversity set
II centered at the Ca2+-binding site in M. tuberculosis GyrA. ................................. 186
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